Long-term goal: The broad objectives of this renewal are to understand the mechanism(s) by which mitochondrial neurotoxins such as 1-methyl-4-phenylpyridinium (MPP+) selectively destroy dopaminergic neurons in the substantia nigra, leading to the development of Parkinson's disease (PD). Reactive oxygen and nitrogen species (ROS/RNS)-mediated damage has been implicated in age-related neurodegenerative diseases like PD. Hypothesis: (i) MPP+ generates mitochondria superoxide (02*) and hydrogen peroxide (H202), and inactivates mitochondrial iron-sulfur-proteins (e.g., aconitase). This stimulates transferrin receptor (TfR)-mediated uptake of iron. (ii) MPP+-induced H202 and iron transported through TfR cause enhanced degradation of tetrahydrobiopterin (BH4), an essential co-factor for neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), and dihydropteridine reductase (DHPR) activities. BH, depletion causes "uncoupling" of nNOS to form O2* and inactivation of TH and DHPR leading to dopamine depletion. (iii) MPP+-induced O2*, H2O2, and Tf-iron stimulate aggregation of a-synuclein, a neuronal presynaptic protein leading to apoptosis or programmed cell death. Aims: 1.) Investigate the effect of TfR-dependent iron and mitochondrial ROS in neuronal cell apoptosis in response to MPP+. 2.) Assess the modulatory effect of BH4 depletion on nNOS-generated nitric oxide ('NO)/O2* ratio and on BH4-dependent enzyme controlling dopamine synthesis. 3.) Elucidate the role of ROS, Tf-iron and BH4 depletion on a-synuclein aggregation and apoptosis in neuronal cells treated with MPP+. Methods: We will use both dopaminergic and non-dopaminergic cells (neuroblastoma and cerebellar granule neurons). The following redox-parameters will be measured: GSH and lipid peroxides; aconitase, complex-I, and iron-regulatory activities; TfR expression and 55Fe uptake; a-synuclein expression and aggregation; caspase activation and apoptosis. ROS/RNS will be determined by fluorescence and spin-trapping techniques. Significance: PD affects about 1% of population over the age of 50. Emerging data allude to environmental mitochondrial toxins as a causative factor. Novelty: This proposal sheds new light on the synergistic role for MPP*-induced mitochondrial ROS, iron, BH4-induced nNOS uncoupling, dopamine depletion and alpha-synuclein aggregation in neuronal toxicity of PD and other mitochondrial diseases.